Polypeptides, cells, and methods involving engineered CD16
This disclosure describes, generally, a modified form of CD 16, genetically-modified cells that express the modified CD 16, and methods that involve the genetically-modified cells. The modified form of CD 16 can exhibit increased anti-tumor and/or anti- viral activity due, at least in part, to reduced susceptibility to ADAM17-mediated shedding upon NK cell stimulation.
1. An isolated mammalian cell or an isolated cell population comprising said isolated mammalian cell, wherein the cell comprises:
a polynucleotide encoding a CD16 polypeptide that comprises: (a) a valine residue at position 196 of SEQ ID NO:1, SEQ ID NO:2, an isoform thereof, or an allelic variant thereof, (b) an amino acid residue other than a serine residue at position 197 of SEQ ID NO:1, SEQ ID NO:2, an isoform thereof, or an allelic variant thereof, and (c) a threonine residue at position 198 of SEQ ID NO:1, SEQ ID NO:2, an isoform thereof, or an allelic variant thereof, wherein the CD16 polypeptide is a functional receptor and has reduced susceptibility to cleavage as compared to a comparable CD16 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, and wherein the amino acid residue other than a serine residue at position 197 is a proline residue.
2. The cell of claim 1 , wherein the cell is a Natural Killer (NK) cell.
3. The cell of claim 2 , wherein the NK cell expresses the CD16 polypeptide, and wherein the cell has reduced CD16 shedding compared to a cell expressing the comparable CD16 polypeptide.
4. The cell of claim 3 , wherein the CD16 polypeptide exhibits reduced susceptibility to cleavage mediated by ADAM17.
5. The cell of claim 1 , wherein the proline residue at position 197 blocks CD16 cleavage.
6. The cell of claim 1 , wherein the proline residue at position 197 results in a conformational change of a cleavage region of CD16 that blocks CD16 cleavage.
7. The cell of claim 2 , wherein the NK cell is a NK cell differentiated from a genetically engineered iPSC or an ESC, wherein the iPSC or ESC comprises the polynucleotide encoding the CD16 polypeptide.
8. The cell of claim 1 , wherein the CD16 polypeptide further comprises a valine residue at position 176 (176V).
9. The cell of claim 2 , wherein the NK cell exhibits at least one of the characteristics selected from the group consisting of:
(a) increased anti-tumor capability;
(b) increased anti-viral capability;
(c) improved antibody-dependent cell cytotoxicity;
(d) increased IFNγ or TNFα production;
(e) increased CD16-mediated activity;
(d) higher surface level of CD16;
(e) lower level of soluble CD16;
(f) enhanced cell stimulation; and
(g) increased in vivo anti-cancer activity, as compared to NK cells expressing the comparable CD16 polypeptide.
10. The cell of claim 7 , wherein the genetically engineered iPSC is a stable iPSC line; or wherein the genetically engineered ESC is a stable ESC line.
11. The cell of claim 7 , wherein the genetically engineered iPSC or ESC is capable of differentiating into genetically engineered hematopoietic cells.
12. The cell of claim 7 , wherein the iPSC or ESC comprising the polynucleotide that encodes the CD16 polypeptide does not express the CD16 polypeptide.
13. The cell of claim 7 , wherein the iPSC or ESC expresses the CD16 polypeptide.
14. The cell of claim 7 , wherein the CD16 polypeptide further comprises a valine residue at position 176 (176V).
15. The cell of claim 7 , wherein the NK cell exhibits at least one of the characteristics selected from the group consisting of:
(a) increased anti-tumor capability;
(b) increased anti-viral capability;
(c) improved antibody-dependent cell cytotoxicity;
(d) increased IFNγ or TNFα production;
(e) increased CD16-mediated activity;
(d) higher surface level of CD16;
(e) lower level of soluble CD16;
(f) enhanced cell stimulation; and
(g) increased in vivo anti-cancer activity, as compared to NK cells expressing the comparable CD16 polypeptide.
16. The cell of claim 1 , wherein the CD16 polypeptide comprises the amino acid sequence of SEQ ID NO:2 except that said amino acid sequence comprises the proline residue at position 197 of SEQ ID NO:2.